The purpose of this project is to investigate the biological and biochemical functions of the HIV accessory proteins Vif and Vpu and to understand their precise role in virus replication and in virus-host interaction. One of our goals is to characterize cellular factors involved in Vif or Vpu function. From our studies on Vpu we expect to gain insights into general principles of protein degradation from the ER and into mechanisms involving late stages of virus production, in particular the involvement of ion channels in the secretory pathway. As a result of our experiments on Vif, we not only expect to gain insights into the function of this viral factor but we expect to learn more about the role of the cytoskeleton in virus replication and general viral assembly mechanisms. Finally, we hope our research will provide a basis for the assessment of viral proteins as potential antiviral targets. The vpu gene is unique to HIV-1 and encodes a small integral membrane protein. Vpu regulates virus release from the cell surface and degradation of CD4 in the endoplasmic reticulum. These two biological activities of Vpu are based on two independent and distinct molecular mechanisms that can be attributed to separable structural domains of Vpu. Vpu-regulated virus release is sensitive to changes in the transmembrane (TM) domain of Vpu and is correlated with an ion channel activity of Vpu. CD4 degradation, on the other hand, involves a direct interaction of the Vpu and CD4 cytoplasmic domains. We demonstrated in the past that Vpu-mediated CD4 degradation involves the ubiquitin-dependent proteasome pathway and requires an interaction with a novel cellular protein, betaTrCP, which is a component of an E3 ubiquitin ligase complex. Unlike normal cellular substrates of TrCP, such as the NF-kappaB inhibitor IkappaB, Vpu is not targeted for degradation by proteasomes. In fact, we found that Vpu has a dominant negative effect on cellular TrCP function by acting as a competitive inhibitor of TrCP. Consistent with its effect on IkappaB-alpha, Vpu substantially repressed the HIV-induced activation of NF-kappaB (Bour et al, 2001). Since NF-kappaB is a key regulator of cytokine expression as well as the expression of anti-apoptotic genes, expression of Vpu in HIV-infected cells was likely to play a role in the perturbation of cytokine production and could be a key factor in HIV pathogenesis. Based on these observations, we have explored in the past year the possible involvement of Vpu in HIV-1-induced apoptosis. We found that in HIV-1-infected CD4+ T cells Vpu contributed significantly to the induction of apoptosis. Using an inducible expression system we found that the effect of Vpu on apoptosis was direct and did not require the coexpression of other viral proteins. Analysis of cellular factors involved in the induction of apoptosis demonstrated that Vpu down-modulated the NF-kappaB-dependent expression of anti-apoptotic genes such as Bcl-xL and A1/Bfl-1. Concomitantly, Vpu expression resulted in increased levels of active caspase-3. These effects of Vpu involved an interaction with TrCP as evidenced by the fact that mutation of the TrCP binding motif in Vpu abolished its apoptogenic potential. These results suggest that Vpu promotes apoptosis through its inhibition of NF-kappaB. Vif is a 23-kDa basic protein, which has an important function in regulating infectivity of progeny virions. Despite the severe impact of Vif defects on virus infectivity, its mechanism of action has thus far remained obscure. It is generally accepted that Vif-deficient viruses can attach to and penetrate host cells but are blocked at a post-penetration step early in the infection cycle. Yet, comparison of virion morphology or protein composition between wild type and Vif-defective virions has thus far been inconclusive and produced conflicting results. Several reports have suggested that Vif affects the stability of the viral nucleoprotein complex. In particular, NC and reverse transcriptase were found to be less stably associated with viral cores in the absence of Vif. Nevertheless, Vif is generally believed to function within the virus-producing cell. This assumption is largely based on the observation that relatively small amounts of Vif seem to be packaged, with estimates ranging from less than 1 to 100 molecules of Vif per virion. Furthermore, packaging of Vif into virus particles is generally believed to be non-specific, leading to questions as to the functional significance of Vif incorporation into virions. In the past year we performed an in-depth biochemical analysis of Vif in purified virions from permissive or restrictive host cells to investigate the specificity of Vif incorporation into virions. Pulse/chase analysis of single-cycle infected H9 cells did not reveal any Vif-dependent differences in viral protein processing and maturation consistent with recent reports by other investigators. Instead, detergent extraction of purified virions demonstrated an association of Vif with the nucleoprotein complex. Interestingly, HIV-1 variants carrying mutations in the nucleocapsid zinc finger domains abolished Vif packaging. In addition, an RNA-packaging defective virus was significantly impaired in packaging of Vif. Finally, deletion of a putative RNA-binding motif between residues 75-114 in Vif abolished its packaging into virions. Taken together, our results indicate that virion packaging of Vif is specific and is mediated by interactions with the viral genomic RNA (Khan et al, 2001).